User:Israr Shah/sandbox1 connexin



Abstract
Heart disease is the leading cause of death for both men and women in the United States. Statistics show that over 80 million people in the United States have some form of cardiovascular disease which is responsible for 1 in every 2.8 deaths. At the molecular level the causes are primarily due to defective connexins.

Connexins are subunits that make up a connexon, a gap junction channel that is involved in cell to cell communication in many organs, and are of crucial importance to the heart. In the heart, connexins are needed for myocardial synchronization and optimal heart function. Any kind of defect or blockage of these connexin channels would therefore lead to interrupted electrical flow and decreased cardiac performance. The most common conditions associated with defective connexin pathways in the heart are arrhythmias or abnormal heart rhythms.

The main connexin involved in intercellular communication is connexin 43 which is abundantly present in the heart, however, the precise function is not clearly understood due to the absence of a known structure. We have generated a three dimensional physical model of a connexon made by connexin 26 subunits, in order to understand specific features of the channel that relates to its isoform. Specifically, we have selected residues that play a key role in permeability, intercellular docking and pore funnel formation. One connexin was selected (blue) to highlight all these features. Residues involved in the intercellular interactions of the two connexons that form the gap junction channels are Leu56, Lys168 , Asp179 , Asn176 and Thr177. Secondary structures in these regions are highlighted and represent beta sheets. The residues involved in formation of the pore funnel are Thr5 and Met34 which interact with Asp2 and Trp3 of an adjacent connexin. It has been observed that Trp3 forms a hydrophobic interaction with Met34, which is thought to keep the channel in an open state. Thr5 forms a hydrogen bond with Asp2. Another group of residues that contribute in regulating the permeability of this channel are Asp 50 and Asp46, which are involved in creating a 9Å long, negatively charged path. This is believed to create selectivity for small, positively charged molecules.

This model will help in future research in heart therapy. If we understand what affects permeability in connexin 43, we can solve many problems related to electrical and molecular coupling such as slow velocity which is involved in arrythmias.

Introduction
Heart disease is the leading cause of death for both men and women in the United States. It is estimated that in 2009, the cost of heart disease will be more than $304.6 billion, including the cost of healthcare and medication (CDC). Statistics show that over 80 million people in the United States have some form of cardiovascular disease, which is responsible for 1 in every 2.8 deaths (American Heart Association). Some common causes of heart disease, especially arrhythmias, include congenital defects, coronary heart disease, high blood pressure, smoking, diabetes, drug abuse and stress. However, on the molecular level the causes are primarily due to defective connexins. Connexins are transmembrane proteins that form gap junctions in vertebrates. Structurally, each gap junction is made up of 2 hemichannels or connexons, which are each made up of 6 connexins. There are more than 20 different types of connexins in the human genome and they are named according to their molecular weights. The connexins that we are focusing on are connexin26 and connexin43. Connexin26 is involved in hearing and defects in this gene are the common cause of congenital deafness. Connexin43 is mainly found in the heart and plays a major role in synchronized contraction of the heart (electrical coupling) and transfer of small molecules (molecular coupling). Mutations in connexin43 result in conditions that cause abnormal electrical activity in the heart which are known as arrhythmias. Ventricular fibrillation, or uncoordinated contraction of the ventricles, is a type of arrhythmia that is the major cause of sudden cardiac death. Its counterpart, Atrial fibrillation, is the most common type of arrhythmia which affects approximately 2.2 million Americans and is responsible for 15% of all stroke cases. The most definitive treatment for fibrillation would be to restore and maintain a normal heart rhythm, in a process called defibrillation. This can be done either by using a defibrillator to give an electrical shock to the heart which resets the heart cells or by anti-arrythmic drugs (AAD’s) which modify the electrical properties of cardiac tissue. Drugs called Gap junction modifying anti-arrythmic peptides (AAP’s) are types of AAD which act by increasing gap junction permeability and can be used in conjunction with defibrillators. Studies have shown that AAP’s, like Rotigaptide, increases the permeability of connexins in the heart and lowers the voltage needed for defibrillation. Other drugs which alter the charge of connexins or somehow be able to constantly dilate the connexin pore will contribute to an increase in permeability. In addition, a drug which works to lower the effective resistance through each connexin or amplify the voltage conductance would also be effective. Therefore, any future insight into the permeability of connexins in the heart would be valuable in areas of heart therapy.

Methods
The PDB for connexin26, 2zw3, was obtained from the RSBS Protein Data Bank website (www. Pdb.org) and the structure of connexin26 was visualized with the computer software RasMol version 2.6.5.1. Using RasMol commands, we selected Beta sheets, Leu56, Lys168, Asp179, Asn176, Thr177, Thr5, Met34, Asp2, Trp3, Asp50 and Asp46. Beta sheets were colored in green, the alpha carbons of Leu56, Lys168, Asp179, Asn176 and Thr177 were colored in orange and Thr5 and Met34 were colored in yellow. Asp2 and Trp3, which interact with Thr5 and Met34 were colored in purple and another set of residues, Asp50 and Asp46 were colored in magenta. After the amino acids were colored, we added monitor lines to wherever they were needed and colored them in CPK. We generated a three dimensional physical model using Rapid Prototyping Machine Zcorp 510printer. We compared the amino acid sequence of connexin26 and connexin43 by using a protein BLAST program. The website used to achieve this was pubmed.org. First we clicked on Blast and then protein blast to paste the sequence of connexin26 in the space provided. Then align two or more sequences was selected and the sequence of connexin43 was pasted in the space provided. To obtain the final results we clicked on BLAST, which is displayed at the bottom of the page.

Results
The amino acid sequences of connexin26 and connexin43 were compared using BLAST program. The two sequences showed 59% positive match. The similarities and differences in amino acids were observed. The amino acids that were similar in both connexin forms were trp3, asp46, asp2, lys168, asn176, and asp179. Meanwhile, the amino acid met34 in connexin 26 was replaced with leucine in connexin 43. Also, the amino acid leucine 65 is present in connexin 26, which is replaced by glutamic acid in connexin 43. The same phenomenon is observed with thr5 and asp50 in connexin 26, which are each replaced by alanine in connexin 43. The final difference is in the amino acid thr177 in connexin 26, which is glu177 in connexin 43.

Discussion
In our research, we used the structure of connexin26 to understand the structure and function of connexin 43 because the structure of connexin 43 was not available to us. The overall structure of connexin26 is formed by two connexons related to each other by a crystallographic two fold symmetry axis. The protomer has four transmembrane segments, two extracellular loops, a cytoplasmic loop, an N-terminal helix and a C – terminal segment. Connexin 26 forms a four helix bundle made up of antiparallel adjacent helices. Transmembrane segments one and two face the interior while transmembrane segments 3 and 4 face the hydrophobic membrane environment. The inner pore lining is made up of transmembrane 1 and 3 which narrow from the cytoplasmic to the extracellular side of the membrane. Permeability in the connexin26 pathway depends on the intracellular channel entrance, a pore funnel and an extracellular cavity. The intracellular entrance of connexin26 is smaller than the entrance of connexin43 and is made up of transmembrane segments 2 and 3. The channel entrance is positively charged which increases the permeability of negatively charges molecules. At the cytoplsmic entrance of the pore, the size and electrical character of the side chains affects molecular size and charge selectivity of the channel. Since the pore diameter is greater in connexin 43, the selectivity is not as strict to small ions and molecules. The inner pore interior contains Asp46 and Asp50 which forms a negatively charged path which, in conjunction with the pore funnel, contributes to size restriction and charge selectivity. Connexin43 shows a lack of selectivity for monovalent ions like potassium, sodium and lithium. BLAST or Basic Local Alignment Search Tool is a program used for comparing primary biological sequence information such as amino acid sequences or nucleotides of different proteins. Comparing the BLAST structures of connexin43 and 26 shows which amino acids are the same and which ones are different. The sequences of connexin43 and connexin26 are 59% similar owing to their similarity in function. Connexin 26 however, has far fewer residues than connexin 43 and this might account for the additional selectivity of Cx26.